Abundance of calpain protein and its activity was increased in AngII-infused aortas

Calpain-1 (A) and spectrin-1 (B,D) protein were detected by Western blotting in tissue extracts from aortas in LDL receptor −/− mice infused with either saline or AngII for 14 days. β-actin was shown as loading control. Calpain-1 (A) and spectrin-1 (B,D) protein abundance was quantified by image analysis. Images are representative out of 4 or 5 independent experiments. Results are represented as means ± SEMs; Calpain activity (C) were measured by fluorimetric assay in aortic tissue extracts from saline and AngII infused mice administered either vehicle or BDA-410 (n=4). Statistical analyses were performed using Students t test (A,B) or two-way ANOVA with a Holm-Sidak multiple comparison post-hoc test (C,D). * and horizontal bars represent significance of P<0.05.

BDA-410 administration did not change calpain-2, cathepsin B and proteasome in AngII-infused aortas

Calpain-2 (A) protein was detected by Western blotting in tissue extracts from aortas in LDL receptor −/− mice infused with either saline or AngII for 14 days. β-actin was shown as loading control. Calpain-2 (A) protein abundance was quantified by image analysis. Images are representative out of 4 independent experiments. Cathepsin B (B) and proteasome activity (C) were measured by fluorimetric assay in aortic tissue extracts from saline and AngII infused mice administered either vehicle or BDA-410 (n=4). Results are represented as means ± SEMs; Statistical analyses were performed using Students t test or two-way ANOVA with a Holm-Sidak multiple comparison post-hoc test. * and horizontal bars represent significance of P<0.05.

Lipoproteins were resolved by size-exclusion chromatography. Total cholesterol concentrations are expressed as mean absorbance per fraction. Symbols represent the means and bars are SEMs of 5 individual mice per group: Vehicle (circles) and BDA-410 (triangles).

Ultrasonic measurements of abdominal aortic diameters were measured on day 0 and after 28 days of AngII-infusion (n=14). Open circles (vehicle) and gray circles (BDA-410) represent individual mice. The length of each box represents the interquartile range (difference between 75th percentile, top of box, and 25th percentile, bottom of box), the dash inside box depicts the median, and the whiskers extending from the top to the bottom of each box indicate the 95th and 5th percentiles, respectively. Statistical analyses were performed using the nonparametric repeated measures of ANOVA on Ranks with Tukey post hoc test. *P<0.05 vs day 0; #P<0.05 vs day 28 BDA-410.

A. Measurements of maximal external width of abdominal aortas (n=14). Open circles (vehicle) and gray circles (BDA-410) represents values from individual mice. The length of each box represents the interquartile range (difference between 75th percentile, top of box, and 25th percentile, bottom of box), the dash inside box depicts the median, and the whiskers extending from the top to the bottom of each box indicate the 95th and 5th percentiles, respectively. Statistical analyses were performed using the nonparametric Mann-Whitney Rank sum test. B. Percent incidence of AAAs based on 50% increase in luminal diameter compared to Day 0. Fisher's exact test was used to determine differences between groups in the incidence of AAA. *P<0.05 BDA-410 vs vehicle.

Atherosclerotic lesion areas were measured on aortic arch intimal surfaces (n = 12–14). Open circles (vehicle) and gray circles (BDA-410) represents values from individual mice. The length of each box represents the interquartile range (difference between 75th percentile, top of box, and 25th percentile, bottom of box), the dash inside box depicts the median, and the whiskers extending from the top to the bottom of each box indicate the 95th and 5th percentiles, respectively. Statistical analyses were performed using the nonparametric Mann-Whitney Rank sum test. *P<0.05 BDA-410 vs vehicle.

Total RNA was extracted and mRNA abundance of MCP-1 (A), IL-6 (B), IL-10 (C), and ICAM-1 (D) genes were analyzed by real-time PCR using β-actin as an internal control (n=4). Values are represented as mean ± SEM. All horizontal bars represent significance of P<0.05 by two-way ANOVA followed by Holm-Sidak post hoc tests.

Total RNA was extracted and mRNA abundance of IKKα (A), IKKβ (B), and IKKε (C) genes were analyzed by real-time PCR using β-actin as an internal control (n=4). Values are represented as mean ± SEM. All horizontal bars represent significance of P<0.05 by two-way ANOVA followed by Holm-Sidak post hoc tests.